Myosin heavy chain (MyHC) is a quintessential marker of skeletal muscle fiber phenotype and function since it is a major determinate of maximum unloaded velocity of shortening (Vmax). Presently, little is known about how the integrative biological response to decreased mechanical loading [hindlimb non-weight bearing (NWB)] leads to fiber-type shifts and altered transcriptional regulation of MyHC genes. Our previous transgenic deletion- mutagenesis and protein-DNA interaction studies identified a negative element (dbetaNRE-S; -332 to -311) that binds two different proteins identified only in NWB-soleus nuclear extract. By affinity chromatography and EMSA analysis we have identified and confirmed that the negative acting single-stranded DNA-binding transcription factors, Puralpha and Purbeta, represent the enriched binding activity we identified in NWB-Soleus nuclear extract. We show that Puralpha and Purbeta decrease betaMyHC reporter gene transcription in muscle cells, and that the dbetaNRE-S element confers Pur-dependent decreased activation of a minimal thymidine kinase promoter. By co- immunoprecipitation we also show that both Pur-proteins interact with Sp3-proteins; transcription factors shown by us to contribute to NWB decreased betaMyHC gene transcription. Since Puralpha and Purbeta have been shown to play a role in decreased cardiac aMyHC and vascular smooth muscle alpha-actin gene transcription, we expect that our findings will have global relevance to the regulation of skeletal, cardiac and smooth muscle gene networks under basal and NWB conditions, and gene regulation in all Pur-protein expressing ceNs. Thus, the primary goal of this proposal is to decipher the physiological role of the Pur-proteins in regulating skeletal muscle phonotype by performing the following aims: 1) to study the phenotypic consequences of transgenic overexpression of Pur- proteins (Puralpha and Purbeta) in adult mouse skeletal muscle, 2) to isolate and study muscle-specific Pur-interacting protein(s) by yeast 2-hybrid and classical proteomics, and 3) to investigate the cooperative interaction between Puralpha, Purbeta, and Sp3-proteins via transient co-transfection assays, chromatin immunoprecipitation with C2C12 myotubes, and GST-interaction assays. Northern and western analysis will assess the impact of Pur-protein overexpression on whole muscle phenotype, which will be correlated to whole muscle and single fiber functional analysis. This work is expected to identify potential protein targets for therapies aimed at providing countermeasures against altered muscle phenotype and debilitating loss of function induced by altered mechanical loads resulting from disease, space flight or extended bed rest.